Immunogenic fusion protein

ABSTRACT

The present invention relates to an immunogenic fusion protein comprising a first amino acid sequence having at least 80% sequence identity with the amino acid sequence of the N-terminal region of a first group B Streptococcus surface protein, which is fused to a second amino acid sequence having at least 80% sequence identity with the amino acid sequence of the N-terminal region of a second group B Streptococcus surface protein. Each of the first and the second group B Streptococcus surface protein is selected from the group consisting of Rib protein, Alp1 protein, Alp2 protein, Alp3 protein, Alp4 protein and AlpC protein. The immunogenic fusion protein further comprises at least one amino acid sequence having at least 80% sequence identity with the amino acid sequence of the N-terminal region of the group B streptococcus surface protein Alp1, Alp2, Alp3 or Alp4. The invention further pertains to an isolated nucleotide sequence encoding the immunogenic fusion protein; a vector; a host cell; an immunogenic product, a vaccine; and a method for preventing or treating a group B Streptococcus infection.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to and is a 35 U.S.C. § 371 nationalphase application of PCT/EP2016/075356 (WO2017/068112), filed on Oct.21, 2016 entitled “IMMUNOGENIC FUSION PROTEIN”, which application claimspriority to and the benefit of Sweden Patent Application No. 1551363-3,filed Oct. 21, 2015 and Sweden Patent Application No. 1551725-3, filedDec. 30, 2015; the disclosures of which are incorporated herein byreference in their entirety.

FIELD OF INVENTION

The present invention relates to the fields of microbiology and vaccinetechnology, and concerns the development of an immunogenic fusionprotein capable of conferring immunity to group B Streptococcusinfections. More particularly, the present invention relates to a novelimmunogenic fusion protein which confers immunity to invasive strains ofthe group B Streptococcus. It further pertains to an isolated nucleotidesequence encoding the immunogenic fusion protein; a vector; a host cell;a vaccine; and a method for preventing or treating a group BStreptococcus infection.

BACKGROUND OF THE INVENTION

Group B Streptococcus (Streptococcus agalactiae) (GBS) is the majorcause of invasive bacterial infections, including meningitis, in theneonatal period. In the United States alone, there are now about 5000cases per year of invasive disease caused by this bacterium. Theseinfections have an overall mortality of about 10%, and many of theinfants that survive have permanent neurological sequelae. In view ofthis, a large effort has been made to find methods of prevention andtreatment and to analyze the mechanisms by which GBS cause infections.

The GBS can also cause mastitis in cows, a bovine disease that is ofconsiderable economical importance. Development of a vaccine against GBSinfections is therefore of interest also in veterinary medicine.

About 20% of all women are vaginal carriers of GBS, and verticaltransmission from the maternal genital tract is probably the most commonsource of infection in neonatal disease caused by this bacterium.However, only about 1% of the infants that are colonized by the GBS atbirth are afflicted by serious infection. Other factors than exposure tothe bacterium during birth must therefore contribute to the developmentof neonatal disease.

Group B streptococcal strains are divided into nine serotypes (Ia, Ib,and II-VIII) based on the structure of the polysaccharide capsule(Baker, J Inf Dis 1990. 161: 917). The four “classical” serotypes Ia,Ib, II, and III occur in roughly equal proportions among strains in thenormal flora, but type III is the clinically most important serotype, inparticular because it causes most cases of meningitis.

Because the capsule is a known virulence factor, it has been studied inconsiderable detail, in particular in type III strains. Efforts havebeen made to develop a vaccine, in which the type III polysaccharidecapsule would be an essential component.

EP 0 866 133 discloses a vaccine capable of protecting a recipient frominfection caused by group B Streptococcus. The invention is directed tothe use of a combination of a polysaccharide and a fragment of theepsilon protein. It further discloses that epidemiological data suggestthat the type-specific capsule plays an important role in the immunityto group B Streptococcus infections (se page 7 line 2-3). Additionally,there are a number of different combinations between different proteinsand the polysaccharide mentioned within the application but all theclaims comprise a polysaccharide which shows the importance of thatparticular component. However, use of the polysaccharide capsule as avaccine may give problems due to cross reactions with human tissues(Pritchard et al., Infect Immun 1992. 60: 1598). It would therefore bevery valuable if one could develop a vaccine based on proteins ratherthan on polysaccharides.

The document Gravekamp et al., Infection and Immunity, December 1997, p5216-5221 discloses the evaluation of the immunogenicity as well asprotection of the number of repeats of the alpha (a) C protein as wellas the N-terminal part alone. It was found that the immunogenicitydecreased with increasing number of repeats (se FIG. 2B). However, itwas also found in a protection assay that the antibodies against theN-terminal region were predominantly responsible for the protectioncompared to antibodies against the N-terminal region (see page 5219 leftcolumn, line 6 from the bottom, and page 5220 right column lines 26-29).

WO 9410317 describes the use of the alpha protein, a GBS surfaceprotein, in the development of a conjugate vaccine. A drawback with thisprotein is that it usually is not expressed by type III strains, whichare the cause of many serious GBS infections. Hence, a protectiveimmunity against these strains will not be evoked by an alpha proteinvaccine.

WO 9421685 describes the use of the Rib protein, a GBS surface protein,in the development of a vaccine. This protein elicits immunity whenadministered with alum. However, the Rib protein has the disadvantagethat it does not evoke a protective immunity against all GBS strains.

WO 2008127179 describes a fusion protein comprising at least one firstN-terminal region fragment of a group B Streptococcus surface protein oranalogue, homologue, derivative or immunologically related amino acidsequence or fragments thereof, which is fused to at least one secondN-terminal region fragment of a group B Streptococcus surface protein oranalogue, homologue, derivative or immunologically related amino acidsequence or fragments thereof, wherein the first and second at least oneN-terminal region fragments of group B Streptococcus surface proteinsderive from different group B Streptococcus strains, and wherein thefusion protein is capable of eliciting protective immunity against groupB Streptococcus.

The document Lindahl et al, Nonimmunodominant Regions Are Effective AsBuilding Blocks In A Streptococcal Fusion Protein Vaccine, Cell Host &Microbe 2, 427-434, December 2007, discloses a fusion protein comprisingN-terminal regions of the group B Streptococcus surface proteins Rib andAlpC.

The document Maeland et al, Survey of Immunological Features of theAlpha-Like Proteins of Streptococcus agalactiae, Clinical and VaccineImmunology, February 2015 Volume 22 Number 2, discloses that atwo-component vaccine, one immunogenic peptide corresponding to a repeatarea stretch of C-alpha or Alp1, either of which cross-reacts strongly(FIG. 2; Table 1), and one peptide corresponding to a repeat areastretch of Alp3 or Rib, either of which also cross-reacts strongly (FIG.3; Table 1), may provide broad protective activity.

Despite the advances in the progress towards a vaccine suitable forprevention of GBS disease, there is still a need for further methods andvaccines for prevention and treatment of GBS infections. Thus, thereremains a need to explore vaccines strategies capable of elicitingprotective immunity against a wide range of GBS stains.

Accordingly, it is a primary objective of the present invention toprovide an immunogenic fusion protein which can be used in a vaccinecapable of eliciting protective immunity against GBS infections.

It is a further objective of the present invention to provide a vaccinethat elicits protective immunity against many clinically important GBSstrains.

Another objective of the present invention is to provide a vaccinecomprising a single, or a few, immunogenic fusion proteins that elicitsprotective immunity against GBS infections. A single or a few proteinshas several advantages over a vaccine composed of numerous proteins,e.g. cost of production and safety.

The means of accomplishing each of the above objectives as well asothers will become apparent from the description of the invention whichfollows hereafter.

SUMMARY OF THE INVENTION

The present invention is based on realization, by the present inventors,that the coverage of the fusion protein disclosed in WO 2008127179 ismore limited than previously thought. This is because it was heretoforethought that the important Group B Streptococcus serotype Ia expressedpredominantly AlpC surface protein, whereas cross reactivity studiescarried out on behalf of the present inventor show that the majority ofthe Group B Streptococcus serotype Ia bacteria express Alp1 instead ofAlpC.

It was furthermore realized that cross-reactivity between differentAlp/Rib N-terminal domains cannot be directly predicted based onsequence homologies between the domains. This was realized when thecross-reactivity of rabbit antibodies raised against the previouslydisclosed Rib-AlpC-NN fusion protein disclosed in WO 2008127179 wastested against N-terminal domains of the Streptococcus Surface proteinsRib, AlpC, Alp1 and Alp2, and against the immunogenic fusion proteinaccording to the first aspect of the present invention as introducedbelow. The results showed similar reactivities against the Rib-AlpC-NNfusion protein and AlpC N-terminal domains. However, 10-fold loverreactivity was observed against Rib and Alp2 N-terminal domains. Evenlower cross-reactivity was observed against Alp1 N-terminal domains, andthe immunogenic fusion protein according to the first aspect of thepresent invention. In addition hereto, mice immunized with theimmunogenic fusion protein according to the first aspect of the presentinvention showed a 2-log reduction in cross-reactivity with theRib-AlpC-NN fusion protein compared to the titer obtained against theimmunogenic fusion protein according to the first aspect of the presentinvention. Likewise, mice immunized with the Rib-AlpC-NN fusion proteinshowed a similar reduction in reactivity against the immunogenic fusionprotein according to the first aspect of the present invention, and inaddition hereto, the maximum amount of binding against thecross-reactive epitopes were also reduced.

Accordingly there was revealed to the present inventors an unexpectedneed for a further immunogenic fusion protein including Alp1, Alp2, orAlp4, for obtaining protection against Group B Streptococcus infections.

Thus a first aspect of the present invention relates to an immunogenicfusion protein comprising:

-   -   a first amino acid sequence having at least 80% sequence        identity with the amino acid sequence of the N-terminal region        of a first group B Streptococcus surface protein, which is fused        to    -   a second amino acid sequence having at least 80% sequence        identity with the amino acid sequence of the N-terminal region        of a second group B Streptococcus surface protein        wherein each of the first and the second group B Streptococcus        surface protein is selected from the group consisting of Rib        protein, Alp1 protein, Alp2 protein, Alp3 protein, Alp4 protein        and AlpC protein, wherein the immunogenic fusion protein        comprises at least one amino acid sequence having at least 80%        sequence identity with the amino acid sequence of the N-terminal        region of the group B streptococcus surface protein Alp1, Alp2,        Alp3 or Alp4, and wherein the immunogenic fusion protein is        capable of eliciting protective immunity against group B        Streptococcus.

A major advantage of the immunogenic fusion protein of the invention isthat it comprises at least one amino acid sequence having at least 80%sequence identity with the amino acid sequence of the N-terminal regionof the group B streptococcus surface protein Alp1, Alp2, Alp3 or Alp4,either of which is expressed by many clinically important strains ofgroup B Streptococcus, and most importantly, it will provide protectiveimmunity against these further important strains.

The immunogenic fusion protein has the advantage that it is immunogeniceven without adjuvant, however it can also be used with an adjuvant forincreased immunogenicity, eliciting protective immunity against strainsexpressing the surface proteins. Moreover, the immunogenic fusionprotein according to the present invention may be used in the vaccineaccording to the present invention and is expected to be administeredwith alum or Aluminium hydroxide (AlOH), an adjuvant accepted for use inhumans. In contrast, the recently described “universal vaccine” was onlyreported to work together with Freund's adjuvant, a strongly irritatingcomponent that cannot be used in human medicine (Maione, D. et al,Science 2005. 309:148-150).

Another advantage with the present invention is that a vaccinecomposition according to the invention can be composed of theimmunogenic fusion protein according to the first aspect of the presentinvention combined with a further fusion protein such as the Rib-AlpC-NNfusion protein of WO 2008127179, thus providing an immunogenic productcapable of providing full coverage of protection against all clinicallyrelevant Group B Streptococcus strains using only two fusion proteinsinstead of needing to use 5 or 6, or even more, different capsularproteins.

More specifically, the present invention relates to the immunogenicfusion protein; an immunogenic product; an isolated nucleotide sequence;a vector; a host cell; a vaccine; and a method for preventing ortreating a group B Streptococcus infection.

The present invention will be described in more detail below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that the Rib-AlpC-NN fusion protein of WO 2008127179provides higher titers of antibodies against the N-terminal regions ofthe homotypic N-terminal domains included in the vaccine antigen, thanit does against the heterotypic cross-reactive N-terminal domains ofAlp1 and Alp2/3. Differences are highlighted by differences infold-increase in FIG. 1C and the larger spread seen between vaccinatedsubjects when looking at the absolute numbers in FIG. 1B,

FIG. 2 shows that a vaccine composition according to the presentinvention composed of an immunogenic fusion protein according to thefirst aspect of the present invention combined with the Rib-AlpC-NNfusion protein of WO 2008127179 provides maximal coverage against allN-terminals, i.e. adding the remaining N-terminal domains to the vaccinecomposition enhances the response to these domains over and above whatis provided in terms of cross-reactivity provided by the Rib-AlpC-NNfusion protein alone,

FIG. 3 shows that there is a linear correlation between IgG levels forthe immunogenic fusion protein of WO 2008127179 and OPA titers againstboth vaccine- and cross-reactive Alp strains, meaning that thosesubjects obtaining high levels of antibodies also against the remainingN-terminal domains these will also be functionally active. Such highlevels are however obtained in fewer subjects when immunized with theRib-AlpC-NN fusion protein alone, hence the inclusion of additionalN-terminal domains in the vaccine composition, and

FIG. 4 shows that alum is a better adjuvant than PolyIC for theimmunogenic fusion proteins according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

In this specification, unless otherwise specified, “a” or “an” means“one or more”.

Throughout the specification, any and all references are specificallyincorporated into this patent application by reference.

In a first embodiment of the immunogenic fusion protein according to thefirst aspect of the present invention the immunogenic fusion proteincomprises:

-   -   a first amino acid sequence having at least 80% sequence        identity with the amino acid sequence of the N-terminal region        of a first group B Streptococcus surface protein, which is fused        to    -   a second amino acid sequence having at least 80% sequence        identity with the amino acid sequence of the N-terminal region        of a second group B Streptococcus surface protein        wherein each of the first and the second group B Streptococcus        surface protein is selected from the group consisting of Rib        protein, Alp1 protein, Alp2 protein, Alp3 protein and AlpC        protein, wherein the immunogenic fusion protein comprises at        least one amino acid sequence having at least 80% sequence        identity with the amino acid sequence of the N-terminal region        of the group B streptococcus surface protein Alp1, Alp2, Alp3 or        Alp4, and wherein the immunogenic fusion protein is capable of        eliciting protective immunity against group B Streptococcus.

The term “immunogenic” is intended to mean having the ability to elicitan immune response. The immunogenic fusion protein of the invention isimmunogenic and characterised by its ability to elicit a protectiveimmune response against at least GBS containing the surface proteins ofwhich the N-terminal regions are comprised by the immunogenic fusionprotein.

For the purpose of the present invention the term “fusion protein”refers to an assembly of two or more protein regions, or fragmentsthereof, comprising for example an N-terminal region of a group BStreptococcus Alp1 protein and an N-terminal region of a group BStreptococcus Alp2 protein. For example there might be one N-terminalregion of the Alp1- and one N-terminal region of the Alp2, or 2, 3, 4 or5 N-terminal region fragments of the Alp1- and the Alp2-proteins,wherein the numbers of N-terminal regions from the two proteins need notbe equal.

The combination of polypeptides to provide a fusion protein can beaccomplished by several means, e.g.: chemically by coupling, conjugationor cross-linking, either directly or through an intermediate structure;physically by coupling through capture in or on a macromolecularstructure; or by molecular biological fusion, through the combination ofrecombinant nucleic acid molecules which comprise fragments of nucleicacid capable of encoding each of the two, such that a single continuousexpression product is finally produced.

For the purpose of the present invention the term “protein” refers to amolecular chain of amino acids. A protein is not of a specific lengthand can, if required, be modified in vivo or in vitro, by, for example,glycosylation, amidation, carboxylation or phosphorylation. Inter alia,peptides, oligopeptides and polypeptides are included within thedefinition. The protein or peptide can be of natural or syntheticorigin. In this context a fusion protein is intended to mean two or morepolypeptides covalently linked to each other either directly orindirectly by several means such as those mentioned above. The term“fused” means to create a fusion protein as mentioned above.

The term “N-terminal region” in relation to the present invention refersto an N-terminus region (N) of a protein. Examples of amino acidsequences of the N-terminal regions of the group B Streptococcus surfaceproteins are given in SEQ IDs NO: 2, 4, 8, 10 and 14.

In particular, examples of N-terminal regions of group B Streptococcusproteins include the N-terminal region of the group B Streptococcus Rib,Alp1, Alp2, Alp3, Alp4 and AlpC protein, including peptides encodingnative amino acid sequences of N-terminal regions of natural Rib, Alp1,Alp2, Alp3, Alp4 and AlpC protein, or may be functional derivatives ofnative sequences of these regions wherein these functional derivativesretain their ability to elicit protective immunity against the group BStreptococcus. The term functional derivatives is intended to includeparts of sequences and fragments of the N-terminal regions; it is alsointended to include variants of the natural proteins (such as proteinshaving changes in amino acid sequence but which retain the ability toelicit an immunogenic, virulence or antigenic property as exhibited bythe natural molecule), for example, with altered flanking sequence.

Group B streptococcal strains, also referred herein as GBS, are wellknown and may be isolated from the blood of infected human beings. GBSis the most common cause of neonatal sepsis in the United States and isresponsible for about 5000 cases per year.

The denotation “Group B streptococcal” derives from the fact thatStreptococci have been divided into immunological groups based upon thepresence of specific carbohydrate antigens on their cell surfaces. Atpresent, groups A through O are recognized (Davis, B. D. et al., In:Microbiology, 3rd. Edition, page 609, (Harper & Row, 1980).

Percent homology can be determined, for example, by comparing sequenceinformation using the GAP computer program, version 6.0, available fromthe University of Wisconsin Genetics Computer Group (UWGCG). The GAPprogram utilizes the alignment method of Needleman and Wunsch (J MolBiol 1970 48:443), as revised by Smith and Waterman (Adv Appl Math 19812:482). Briefly, the GAP program defines similarity as the number ofaligned symbols (i.e., nucleotides or amino acids) which are similar,divided by the total number of symbols in the shorter of the twosequences. The preferred default parameters for the GAP program include:(1) a unitary comparison matrix (containing a value of 1 for identitiesand 0 for non-identities) and the weighted comparison matrix of Gribskovand Burgess (Nucl Acids Res 1986 14:6745), as described by Schwartz andDayhoff, eds. (Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, Washington, D.C. 1979, pp. 353-358); (2)a penalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps.

The group B Streptococcus Rib protein, also referred to in thisspecification as Rib and Rib protein, is a surface protein known in theart, and for example described in WO 9421685. The denotation “Rib”refers to: Resistance to proteases, immunity, and group B. The Ribprotein was first isolated from a group B streptococcal strain ofserotype III as a distinct 95 kDa protein. Protein Rib is expressed byalmost all group B streptococcal strains of the clinically importantserotype III, which cause most cases of meningitis, and by some strainsof other serotypes such as II. Moreover, Rib is expressed by all strainsof a hypervirulent clone of type III. A method has been devised topurify protein Rib and it has been demonstrated that antibodies to thisprotein protect against lethal infection with strains expressing proteinRib (for further details, such as DNA and protein sequences see WO9421685). The nucleic acid sequence and the amino acid sequence for theN-terminal region of Rib are given in SEQ ID Nos: 1 and 2.

The Alp1 protein is also known as epsilon protein and is a group Bstreptococcal alpha-protein-like protein (Creti et al. Clin Microbiol.2004.42:1326-9).

The nucleic acid sequence and the amino acid sequence for the N-terminalregion of Alp1 are given in SEQ ID Nos: 7 and 8. The amino acid sequenceis (SEQ ID No 8):

MAEVISGSAATLNSALVKNVSGGKAYIDIYDVKNGKIDPLNLIVLTPSNYSANYYIKQGGRIFTSVNQLQTPGTATITYNILDENGNPYTKSDGQIDIVSLVTTVYDTTELRNNINKVIENANDPKWSDDSRKDVLSKIEVIKNDIDNNPKTQSDIDNKIVEVNELEKLLVLP

The Alp2 protein is another alpha-protein-like-protein first identifiedin a serotype V strain (Lachenauer, C. S., R. Creti, J. L. Michel, andL. C. Madoff. 2000. Mosa-icism in the alpha-like protein genes of groupB streptococci. Proc. Natl. Acad. Sci. USA 97:9630-9635.). Like theother members of the family, the Alp2 protein has an N-terminal domainand several repeated domains towards the C-terminus. Subsequently thatprotein has been found also in other GBS isolates such as serotypes Iaand III (Lindahl et al. Surface Proteins of Streptococcus agalactiae andRelated Proteins in Other Bacterial Pathogens, CLINICAL MICROBIOLOGYREVIEWS, January 2005, p. 102-127). The nucleic acid sequence and theamino acid sequence for the N-terminal region of Alp2 are given in SEQID Nos: 9 and 10.

The Alp3 protein is yet another alpha-protein-like-protein, also know asR28. It is very similar to the R28 protein also found in S. pyrogenes.(Lachenauer, C. S., R. Creti, J. L. Michel, and L. C. Madoff. 2000.Mosa-icism in the alpha-like protein genes of group B streptococci.Proc. Natl. Acad. Sci. USA 97:9630-9635 and Lindahl et al. SurfaceProteins of Streptococcus agalactiae and Related Proteins in OtherBacterial Pathogens, CLINICAL MICROBIOLOGY REVIEWS, January 2005, p.102-127). The structure is more complex than the otherAlpha-protein-like-proteins, but it retains an N-terminal domain whichis identical to that of Alp2, and C-terminal repeat regions very similarto Rib. The nucleic acid sequence and the amino acid sequence for theN-terminal region of Alp3 are the same as for Alp2 and are given in SEQID Nos: 9 and 10.

The Alp4 protein is an alpha-protein-like-protein so far only identifiedin the Prague 25/60 strain (Fanrong Kong, Sonia Gowan, Diana Martin,Gregory James, and Gwendolyn L. Gilbert. Molecular Profiles of Group BStreptococcal Surface Protein Antigen Genes: Relationship to MolecularSerotypes. JOURNAL OF CLINICAL MICROBIOLOGY, February 2002, p. 620-626).It is a novel member of the Alpha-protein-like family with a structuresimilar to that of the other members, with a distinct N-terminal domain,and repeat regions towards the C-terminus.

The nucleic acid sequence and the amino acid sequence for the N-terminalregion of Alp4 are given in SEQ ID Nos 13 and 14.

The group B Streptococcus AlpC protein, also known as alpha protein, isa group B Streptococcus surface protein known in the art. WO 9410317describes a conjugate vaccine composition comprising the alpha protein.The native group B Streptococcus AlpC precursor protein as described inWO 9410317 has a molecular weight of 108 kDa. Cleavage of the putativesignal sequence of 41 amino acids yields a mature protein of 104 kDa.(Note, however, that the signal sequence was subsequently shown to havea length of 56 amino acid residues: Stålhammar-Carlemalm et al., J ExpMed 177, 1593; 1993). The 20 kDa N-terminal region of the AlpC antigenshows no homology to previously described protein sequences and isfollowed by a series of nine tandem repeating units that make up 74% ofthe mature protein. Each repeating unit (denoted herein as “R”) isidentical and consists of 82 amino acids with a molecular mass of about8500 Daltons, which is encoded by 246 nucleotides. The C-terminal regionof the AlpC antigen contains a cell wall anchor domain motif present ina number of Gram-positive surface proteins.

The nucleic acid sequence and the amino acid sequence for the N-terminalregion of AlpC are given in SEQ ID Nos: 3 and 4.

Each of the Rib, Alp1, and AlpC proteins of GBS includes a uniqueN-terminal region (N) and a long repeat (R) region. The proteinsexpressed by the GBS strains BM110 and A909 have 12 and 9 repeats,respectively. The wall anchoring regions are located at the C-terminalends.

The N-terminal regions of Alp2 and Alp3 are identical.

The tandem repeats in Rib and alpha are identical within each protein,but not between the proteins, and vary in number between isolates.Except for this variation, the sequences of Rib and alpha are stableamong strains. The two proteins show little or no antigeniccross-reactivity.

The term “protective immunity” in relation to the present inventionrefers to the ability of serum antibodies and/or cytotoxic T cellresponse induced during immunization to protect (partially or totally)against disease caused by an infectious agent, such as a group BStreptococcus. That is, a vertebrate immunized by the vaccines of theinvention will experience limited growth and spread of group BStreptococcus. To determine whether protective immunity is induced by afusion protein or vaccine, techniques well known for a person skilled inthe art can be used. For example, to determine whether immunization witha fusion protein or vaccine of the invention induces protective immunityagainst group B Streptococcus infection, immunized test animals can bechallenged with group B Streptococcus and growth and spread of the groupB Streptococcus is measured. For example to determine whether protectiveimmunity is induced, methods in accordance with the methods described inthe examples below can be used.

In one embodiment of the immunogenic fusion protein according to thefirst aspect of the present invention the first amino acid sequence mayhave at least 90 such as 95, 96, 97, 98 or 99% sequence identity withthe amino acid sequence of the N-terminal region of the first group BStreptococcus surface protein, and the second amino acid sequence mayhave at least 90, such as at least 95, 96, 97, 98 or 99% sequenceidentity with the amino acid sequence of the N-terminal region of thesecond group B Streptococcus surface protein.

The immunogenic fusion protein comprises at least one amino acidsequence having at least 80%, such as at least 90 such as 95, 96, 97,98, or 99% sequence identity with the amino acid sequence of theN-terminal region of the group B streptococcus surface protein Alp1,Alp2, Alp3 or Alp4.

In one preferred embodiment of the immunogenic fusion protein accordingto the first aspect of the present invention the immunogenic fusionprotein comprises at least one amino acid sequence having at least 80%,such as at least 90 such as 95, 96, 97, 98, or 99% sequence identitywith the amino acid sequence of the N-terminal region of the group Bstreptococcus surface protein Alp1, Alp2 or Alp3.

In a more preferred embodiment of the immunogenic fusion proteinaccording to the first aspect of the present invention the immunogenicfusion protein comprises at least one amino acid sequence having atleast 80%, such as at least 90 such as 95, 96, 97, 98, or 99% sequenceidentity with the amino acid sequence of the N-terminal region of thegroup B streptococcus surface protein Alp1.

In a further embodiment of the immunogenic fusion protein according tothe first aspect of the present invention the immunogenic fusion proteinfurther comprises

-   -   a third amino acid sequence having at least 80% sequence        identity with the amino acid sequence of the N-terminal region        of a third group B Streptococcus surface protein, which is fused        to one of the first and second amino acid sequences, the third        group B Streptococcus surface protein being selected from the        group consisting of Rib protein, Alp1 protein, Alp2 protein,        Alp3 protein, Alp4 protein and AlpC protein,

This is advantageous as it provides for an immunogenic fusion proteincapable of eliciting protective immunity against a larger number ofgroup B Streptococcus strains.

In one embodiment of the immunogenic fusion protein according to thefirst aspect of the present invention the third amino acid sequence hasat least 90 such as at least 95, 96, 97, 98 or 99% sequence identitywith the amino acid sequence of the N-terminal region of the third groupB Streptococcus surface protein.

In a further embodiment of the immunogenic fusion protein according tothe first aspect of the present invention the immunogenic fusion proteinfurther comprises a fourth amino acid sequence having at least 80%sequence identity with the amino acid sequence of the N-terminal regionof a fourth group B Streptococcus surface protein, which is fused to oneof the first and third amino acid sequences, the fourth group BStreptococcus surface protein being selected from the group consistingof Rib protein, Alp1 protein, Alp2 protein, Alp3 protein, Alp4 proteinand AlpC protein.

This is advantageous as it provides for an immunogenic fusion proteincapable of eliciting protective immunity against a larger number ofgroup B Streptococcus strains.

In one embodiment of the immunogenic fusion protein according to thefirst aspect of the present invention the fourth amino acid sequence hasat least 90 such as at least 95, 96, 97, 98 or 99% sequence identitywith the amino acid sequence of the N-terminal region of the fourthgroup B Streptococcus surface protein.

It should be emphasized that the first, second, third and fourth aminoacid sequences may be arranged in any order in the immunogenic fusionprotein. That being said the amino acid sequences are preferablyarranged from first to fourth.

In one embodiment of the immunogenic fusion protein according to thefirst aspect of the present invention the immunogenic fusion proteinconsists of the first and the second amino acids sequences, the first,second and third amino acid sequences, or alternatively the first,second, third and fourth amino acid sequences.

In a further embodiment of the immunogenic fusion protein according tothe first aspect of the present invention the first and second group BStreptococcus surface proteins, or optionally the first, second andthird group B Streptococcus surface proteins, or optionally the first,second, third and fourth group B Streptococcus surface proteins, arederived from different group B Streptococcus strains.

This will imply slight variability in the sequence of the N-terminalregion fragments but would not alter the biological properties and theirfunctional ability to elicit protective immunity. This is advantageousas it increases the number of group B Streptococcus strains which theimmunogenic fusion protein according to the first aspect of the presentinvention provides protection against.

In a further embodiment of the immunogenic fusion protein according tothe first aspect of the present invention the first and second group BStreptococcus surface proteins, or optionally the first, second andthird group B Streptococcus surface proteins, or optionally the first,second, third and fourth group B Streptococcus surface proteins, aredifferent.

This is advantageous as it increases the number of group B Streptococcusstrains which the immunogenic fusion protein according to the firstaspect of the present invention provides protection against.

In a further embodiment of the immunogenic fusion protein according tothe first aspect of the present invention one of the first and secondgroup B Streptococcus surface proteins is Alp1 protein and the other isAlp2 protein, or vice versa.

This is advantageous as it is expected that the fusion of theN-terminals of Alp1 and Alp2 will provide benefits in protecting againstgroup B Streptococcus.

In a further embodiment of the immunogenic fusion protein according tothe first aspect of the present invention the first amino acid sequencehas at least 80%, such as at least 85%, such as at least 90%, such as95, 96, 97, 98 or 99% sequence identity with the amino acid sequence SEQID NO:8, and

-   -   the second amino acid sequence has at least 80%, such as at        least 85%, such as at least 90%, such as 95, 96, 97, 98 or 99%        sequence identity with the amino acid sequence SEQ ID NO:10.

The term “sequence identity” indicates a quantitative measure of thedegree of homology between two amino acid sequences of equal length orbetween two nucleotide sequences of equal length. If the two sequencesto be compared are not of equal length, they must be aligned to bestpossible fit. Sequence identity can, for example, be calculated by theBLAST program e.g. the BLASTP program or the BLASTN program (Pearson W.R and D. J. Lipman (1988) PNAS USA 85:2444-2448)(www.ncbl.nlm.nlh.gov/BLAST).

In a further embodiment of the immunogenic fusion protein according tothe first aspect of the present invention the immunogenic fusion proteincomprises an amino acid sequence having at least 80%, such as at least85%, such as at least 90%, such as 95, 96, 97, 98 or 99% sequenceidentity with the amino acid sequence shown in SEQ ID NO:12.

SEQ ID NO: 12 shows the amino acid sequence of an immunogenic fusionprotein comprising the N-terminal of Alp1 fused to the N-terminal ofAlp2. The amino acid sequence (SEQ ID NO: 12) is:

MAEVISGSAATLNSALVKNVSGGKAYIDIYDVKNGKIDPLNLIVLTPSNYSANYYIKQGGRIFTSVNQLQTPGTATITYNILDENGNPYTKSDGQIDIVSLVTTVYDTTELRNNINKVIENANDPKWSDDSRKDVLSKIEVIKNDIDNNPKTQSDIDNKIVEVNELEKLLVLPEFSTIPGSAATLNTSITKNIQNGNAYIDLYDVKNGLIDPQNLIVLNPSSYSANYYIKQGAKYYSNPSEITTTGSATITFNILDETGNPHKKADGQIDIVSVNLTIYDSTALRNRIDEVINNANDPKWSDGSRDEVLTGLEKIKKDIDNNPKTQIDIDNKINEVNEIEKLLVVSL

In a further embodiment of the immunogenic fusion protein according tothe first aspect of the present invention the immunogenic fusion proteinis modified by glycosylation, amidation, carboxylation orphosphorylation, or by being conjugated to a capsular polysaccharide oran RSV antigen as described with regard to the third aspect of thepresent invention further below.

This is advantageous as such polypeptides may have enhancedimmunogenicity. Such polypeptides may result when the native forms ofthe polypeptides or fragments thereof are modified or subjected totreatments to enhance their immunogenic character in the intendedrecipient. Numerous techniques are available and well known to those ofskill in the art which may be used, without undue experimentation, tosubstantially increase the immunogenicity of the polypeptides hereindisclosed. For example, the polypeptides may be modified by coupling todinitrophenol groups or arsanilic acid, or by denaturation with heatand/or SDS. Particularly if the polypeptides are small polypeptidessynthesized chemically, it may be desirable to couple them to animmunogenic carrier. The coupling of course, must not interfere with theability of either the polypeptide or the carrier to functionappropriately. For a review of some general considerations in couplingstrategies, see Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, ed. E. Harlow and D. Lane (1988). Useful immunogeniccarriers are well known in the art. Examples of such carriers arekeyhole limpet hemocyanin (KLH); albumins such as bovine serum albumin(BSA) and ovalbumin, PPD (purified protein derivative of tuberculin);red blood cells; tetanus toxoid; cholera toxoid; agarose beads;activated carbon; or bentonite.

A second aspect of the present invention concerns an immunogenic productcomprising the immunogenic fusion protein according to the first aspectof the present invention and further comprising a second immunogenicprotein comprising at least two amino acid sequences, wherein the twoamino acid sequences consists of a first amino acid sequence having atleast 80%, such as at least 85%, such as at least 90%, such as 95, 96,97, 98 or 99% sequence identity with an amino acid sequence as shown inSEQ ID NO:2, fused to a second amino acid sequence having at least 80%,such as at least 85%, such as at least 90%, such as 95, 96, 97, 98 or99% sequence identity with an amino acid sequence as shown in SEQ IDNO:4, wherein the second immunogenic fusion protein is capable ofeliciting protective immunity against group B Streptococcus.

The second immunogenic fusion protein may have the amino acid sequenceshown in SEQ ID NO: 6. A corresponding DNA sequence is shown in SEQ IDNO: 5. This is advantageous as it provides an immunogenic productcapable of providing full coverage of protection against all clinicallyrelevant Group B Streptococcus strains using only two fusion proteinsinstead of needing to use 5 or 6, or even more, different capsularproteins.

Preferably the immunogenic product according to the second aspect of thepresent invention comprises the immunogenic fusion protein according tothe first aspect of the present invention wherein one of the first andsecond group B Streptococcus surface proteins (in the immunogenic fusionprotein) is Alp1 protein and the other is Alp2 protein. More preferablythe first amino acid sequence (in the immunogenic fusion protein) has atleast 80%, such as at least 85%, such as at least 90%, such as 95, 96,97, 98 or 99% sequence identity with the amino acid sequence SEQ IDNO:8, and the second amino acid sequence (in the immunogenic fusionprotein) has at least 80%, such as at least 85%, such as at least 90%,such as 95, 96, 97, 98 or 99% sequence identity with the amino acidsequence SEQ ID NO:10. Even more preferably the immunogenic fusionprotein comprises an amino acid sequence having at least 80%, such as atleast 85%, such as at least 90%, such as 95, 96, 97, 98 or 99% sequenceidentity with the amino acid sequence shown in SEQ ID NO:12.

Thus in one preferred embodiment of the immunogenic product according tothe second aspect of the present invention the immunogenic fusionprotein comprises the amino acid sequence shown in SEQ ID NO:12, and thesecond immunogenic fusion protein comprises the amino acid sequenceshown in SEQ ID NO: 6.

Further, in one embodiment of the immunogenic product according to thesecond aspect of the present invention the immunogenic fusion proteinconsists of the amino acid sequence shown in SEQ ID NO:12 and the secondimmunogenic fusion protein consists of the amino acid sequence shown inSEQ ID NO: 6. Preferably there are no other proteins or amino acidssequences, different from the amino acid sequences in SEQ ID NO:6 and12, in the immunogenic product.

FIG. 1A shows that the Rib-AlpC-NN fusion protein of WO 2008127179provides higher titers of antibodies against the N-terminal regions ofthe homotypic N-terminal domains included in the vaccine antigen, thanit does against the heterotypic cross-reactive N-terminal domains ofAlp1 and Alp2/3. Differences are highlighted by differences infold-increase in FIG. 1C and the larger spread seen between vaccinatedsubjects when looking at the absolute numbers in FIG. 1B. The effect ofthe immunogenic product according to the second aspect of the presentinvention is further shown in FIG. 2A-C. Thus FIG. 2A shows thatimmunization with a RibN-AlpCN fusion protein of WO 2008127179 (referredto as GBS-NN) provides equal titers against the N-terminal regions orRib and AlpC, but less cross-reactive titers. On the other handimmunization with an Alp1N-Alp2/3N fusion protein according to the firstaspect of the present invention (referred to as GBS-NN2) provides equaltiters against the N-terminal regions of Alp1 and Alp2/3, however alsohere with less cross-reactive titers, as seen in FIG. 2B. When the twofusion proteins are combined in the immunogenic product according to thesecond aspect of the present invention (referred to as GBS-NN+NN2) as inFIG. 2C, the coverage (titers) is increased for all N-terminal regionsof the Rib, AlpC, Alp1, Alp2/3. Accordingly, a very broad protectionagainst GBS is achieved by the immunogenic product according to thesecond aspect of the present invention.

Further, FIG. 3 shows that that there is a linear correlation betweenIgG levels for the RibN-AlpCN immunogenic fusion protein of WO2008127179 and OPA titers against both vaccine- and cross-reactive Alpstrains. Thus FIG. 3 shows that antibodies generated against theRibN-AlpCN immunogenic fusion protein, specific for the N-terminals orRib and AlpC found in the fusion protein as well as specific for theother N-terminal regions, i.e. the N-terminal regions for Alp1 andAlp2/3 (for Alp1N and alp2/3N by cross-reactivity) are functionallyactive and can kill GBS bacteria with opsono-phagocytosis. This meansthat those subjects obtaining high levels of antibodies also against theremaining N-terminal domains these will also be functionally active.Such high levels are however obtained in fewer subjects when immunizedwith the Rib-AlpC-NN fusion protein alone, hence the inclusion ofadditional N-terminal domains, i.e. the immunogenic fusion proteinaccording to the first aspect of the present invention, in theimmunogenic product and/or the vaccine.

A third aspect of the present invention concerns a vaccine comprising apharmaceutically acceptable vehicle, optionally an adjuvant, and apharmaceutically effective amount of an immunogenic fusion proteinaccording to the first aspect of the present invention or an immunogenicproduct according to the second aspect of the present invention whereinthe vaccine is capable of eliciting protective immunity against group BStreptococcus.

The term “pharmaceutical acceptable vehicle” is intended to mean anysuitable acceptable excipient, adjuvants, carrier, diluent commonly usedin pharmaceutical formulations.

The vaccine may be a vaccine composition.

The vaccine may, in addition to the fusion protein, comprise otherpharmacologically acceptable ingredients such as salts, buffers,immunoactive components, adjuvants (AlOH), wetting agents, emulsifyingand suspending agents, or sweetening, flavouring, perfuming agents, orother substances which are desirable for improving the efficacy of thecomposition. A composition is said to be “pharmacologically acceptable”if its administration can be tolerated by a recipient individual.

In a preferred embodiment of the vaccine according to the third aspectof the present invention the vaccine comprises a pharmaceuticallyeffective amount of the immunogenic product according to the secondaspect of the present invention.

This is advantageous as the immunogenic product provides broadprotection against GBS.

A multivalent vaccine may also be prepared by combining the immunogenicfusion protein or the immunogenic product with other components,including other fusion proteins as described above, including but notlimited to diphtheria toxoid or tetanus toxoid, or polysaccharides,using techniques known in the art. The vaccine may further comprisefurther antigens such as RSV antigens or E. coli antigens.

Methods for the preparation and formulation of vaccines and vaccinecompositions are well known to those skilled in the art. The choice ofingredients will for instance vary depending on the administration routeof the composition. For example compositions for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Carriers orocclusive dressings can be used to increase skin permeability andenhance antigen absorption. Liquid dosage forms for oral administrationmay generally comprise a liposome solution containing the liquid dosageform. Suitable forms for suspending liposomes include emulsions,suspensions, solutions, syrups, and elixirs containing inert diluentscommonly used in the art, such as purified water.

In a further embodiment of the third aspect of the present invention thevaccine may comprise an additional immunoactive component. Theadditional immunoactive component may be an antigen, an immune enhancingsubstance, and/or a vaccine; either of these may comprise an adjuvant.

Adjuvants are substances that can be used to specifically augment aspecific immune response. Normally, the adjuvant and the composition aremixed prior to presentation to the immune system, or presentedseparately, but into the same site of the animal or human beingimmunized. Adjuvants can be loosely divided into several groups basedupon their composition. These groups include oil adjuvants (for example,Freund's complete and incomplete), mineral salts for example, AlK(SO₄)₂, AlNa (SO₄)₂, AlNH₄ (SO₄), AlOH, silica, kaolin, and carbon),polynucleotides (for example, poly IC and poly AU acids), and certainnatural substances (for example, wax D from Mycobacterium tuberculosis,as well as substances found in Corynebacterium parvum, or Bordetellapertussis, and members of the genus Brucella. Among those substancesparticularly useful as adjuvants are saponins such as, for example, QuilA. Examples of materials suitable for use in vaccine compositions areprovided in Remington's Pharmaceutical Sciences (Osol, A, Ed, MackPublishing Co, Easton, Pa., pp. 1324-1341 (1980).

The impact of two different adjuvants on the immune response of thepreviously described fusion protein of WO 2008127179 has been tested inmice. The mice were immunized once at day 0 without adjuvant, or withAlhydrogel (aluminium hydroxide) or PolyIC as an adjuvant. At week 15,significantly higher titers were seen in mice immunized in the presenceof Alhydrogel (approx. 5×10³), compared both to without adjuvant(approx. 2×10¹) and with PolyIC (approx. 10²).

Challenging the mice at week 15 with protein without adjuvants, andmeasuring at antibody titers at week 16, had relatively little effect onthe group originally receiving protein without adjuvant (A), a 1-logincrease on the PolyIC group (C), but relatively little effect on thealready high Alhydrogel group (B). Even though the titers in the PolyICgroup did increase it did not reach the level of the Alhydrogel group.

Thus it is expected that Alhydrogel will be an especially effectiveadjuvant for the immunogenic fusion protein according to the firstaspect of the present invention and therefore especially useful in thevaccine according to the third aspect of the present invention.

The results are shown in FIG. 4. The mice were immunized with 3 μg ofthe RibN-AlpC-N (referred to as GBS-NN) fusion protein of WO 2008127179.

Accordingly, in a preferred embodiment of the vaccine according to thethird aspect of the present invention the vaccine further comprisesaluminium hydroxide as an adjuvant.

Further, in one embodiment of the vaccine according to the third aspectof the present invention the vaccine consists of a pharmaceuticallyeffective vehicle, aluminium hydroxide, and the immunogenic product ofaccording to the second aspect of the present invention, wherein, in theimmunogenic product according to the second aspect of the presentinvention, the immunogenic fusion protein consists of the amino acidsequence shown in SEQ ID NO:12 and the second immunogenic fusion proteinconsists of the amino acid sequence shown in SEQ ID NO 6. Preferablythere are no other proteins or amino acids sequences, different from theamino acid sequences in SEQ ID NO:6 and 12, in the vaccine.

In a further embodiment of the third aspect of the present invention thevaccine, alternatively or further, comprises a host cell according tothe seventh aspect of the present invention

In a further embodiment of the third aspect of the present invention theimmunogenic fusion protein is conjugated to a capsular polysaccharide,preferably a bacterial polysaccharide, more preferably a group BStreptococcus polysaccharide. The use of a polypeptide, protein orfusion protein as a carrier for a polysaccharide in a conjugate vaccineis well known in the art, see for example U.S. Pat. No. 6,855,321, WO9410317 and U.S. Pat. No. 4,496,538).

By polysaccharide is meant any linear or branched polymer consisting ofmonosaccharide residues, usually linked by glycosidic linkages, and thusincludes oligosaccharides. Preferably, the polysaccharide will containbetween 2 and 50 monosaccharide unites, more preferably between 6 and 30monosaccharide units. The polysaccharide component may be based on orderived from polysaccharide components of the polysaccharide capsulefrom many Gram positive and Gram negative bacterial pathogens such as H.influenzae, N. meningitidis and S. pneumoniae. Other bacteria from whichpolysaccharide components may be conjugated to the carrier proteins ofthe present invention include Staphylococcus aureus, Klebsiella,Pseudomonas, Salmonella typhi, Pseudomonas aeruginosa, and Shigelladysenteriae. Polysaccharide components suitable for use according tothis aspect of the present invention include the Hib oligosaccharide,lipopolysaccharide from Pseudomonas aeruginosa (Seid and Sadoff, 1981),lipopolysaccharides from Salmonella (Konadu et al., 1996) and theO-specific polysaccharide from Shigella dysenteriae (Chu et al, 1991).Other polysaccharide components suitable for use in accordance with thepresent invention will be well-known to those skilled in the art.Fragments of bacterial capsular polysaccharide may be produced by anysuitable method, such as by acid hydrolysis or ultrasonic irradiation(Szn et al, 1986). Other methods of preparation of the polysaccharidecomponents will be well known to those of skill in the art.

In one embodiment of the present invention, the polysaccharide is acapsular polysaccharide derived from group B Streptococcus, or theirequivalents.

The polysaccharide component of the conjugate vaccine should preferablybe coupled to the carrier protein by a covalent linkage. A particularlypreferred method of coupling polysaccharide and protein is by reductiveamination. Other methods include: activation of the polysaccharide withcyanogen bromide followed by reaction with adipic acid dihydrazide(spacer) and by conjugation to carboxide groups of carrier protein usingsoluble carbodiimides (Shneerson et al, 1986); functionalisation of thecarrier protein with adipic acid dihydrazide followed by coupling tocyanogen bromide activated polysaccharides (Dick et al, 1989); chemicalmodification of both the carrier protein and the polysaccharide followedby their coupling (Marburg et at, 1986; Marburg et al, 1987 and 1989).

The polysaccharide molecule may be coupled to the carrier protein by aspacer molecule, such as adipic acid. This spacer molecule can be usedto facilitate the coupling of protein to polysaccharide. After thecoupling reaction has been performed, the conjugate may be purified bydiafiltration or other known methods to remove unreacted protein orpolysaccharide components.

If the polysaccharide is derived from a bacterial pathogen differentfrom GBS, the conjugate may elicit immunity against two or morepathogens, e.g. multiple types of bacteria. This is a potentiallyimportant application of the immunogenic fusion protein. For thepreparation of a conjugate vaccine, it would be a considerable advantagethat the protein part is composed of a single fusion protein.

It is apparent to an artisan of skill in the art that vaccinecomposition of the present invention may comprise other substances orcompounds not mentioned above, such as other diluents, emulsifying orstabilizing agents, or other proteins or polysaccharides. Suchsubstances or compounds should confer desired properties to thecomposition.

The vaccine according to the third aspect of the present invention maybe administrated parenterally, intramuscularly, intravenously,intraperitoneally, intradermally, mucosally, submucosally, topically orsubcutaneously.

A fourth aspect of the present invention concerns the vaccine accordingto the third aspect of the present invention for use in preventing ortreating an infection caused by a group B Streptococcus.

A fifth aspect of the present invention concerns a nucleotide sequencecomprising

-   -   at least one first nucleotide sequence as shown in SEQ ID NO:7        or fragments thereof fused, for example by being connected        chemically, by being conjugated, or by being cross-linked, to    -   at least one second nucleotide sequence as shown in SEQ ID NO:9        or fragments thereof, or alternatively,    -   at least one nucleotide sequence as shown in SEQ ID NO:11.

A sixth aspect of the present invention concerns a vector comprising thenucleotide sequence according to the fifth aspect of the presentinvention.

A wide variety of expression host/vector combinations may be employed inexpressing the nucleotide sequences of this invention. Useful expressionvectors for eukaryotic hosts include, for example, vectors comprisingexpression control sequences from SV40, bovine papilloma virus,adenovirus, adeno-associated virus, cytomegalovirus, and retroviruses.Useful expression vectors for bacterial hosts include bacterialplasmids, such as those from E. coli, including pBluescript, pGEX2T, pUCvectors, col E1, pCR1, pBR322, pMB9 and their derivatives, wider hostrange plasmids, such as RP4, phage DNAs, e.g., the numerous derivativesof phage lambda, e.g., lambda GT10 and lambda GT11, NM989, and other DNAphages, such as M13 and filamentous single stranded DNA phages. Usefulexpression vectors for yeast cells include the 2.mu. plasmid andderivatives thereof. Useful vectors for insect cells include pVL 941.

In addition, any of a wide variety of expression control sequences maybe used in these vectors to express the nucleotide sequences/DNAsequences of this invention. Useful expression control sequences includethe expression control sequences associated with structural genes of theforegoing expression vectors. Examples of useful expression controlsequences include, for example, the early and late promoters of SV40 oradenovirus, the lac system, the trp system, the TAC or TRC system, theT3 and T7 promoters, the major operator and promoter regions of phagelambda, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., Pho5, the promoters of the yeast alpha-matingsystem and other constitutive and inducible promoter sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

A seventh aspect of the present invention concerns a host cellcomprising the vector according to the sixth aspect of the presentinvention.

In one embodiment of the seventh aspect of the present invention thehost cell may be a gram negative bacterial cell, gram positive bacterialcell, yeast cell, insect cell, animal cell, African green monkey cell,human cell, or plant cell.

In further embodiments of the host cell according to the seventh aspectof the present invention the host cell may be selected from the groupconsisting of E. coli, Pseudomonas, Bacillus, Streptomyces, aspergillus,lactobacillus, shigella, salmonella, listeria, streptococcus,staphylococcus and fungi. E. coli is however preferred.

A wide variety of unicellular host cells are useful in expressing thenucleotide sequences/DNA sequences of this invention. These hosts mayinclude well known eukaryotic and prokaryotic hosts, such as both gramnegative and gram positive strains, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, streptococcus, staphylococcus,lactobacillus, aspergillus, shigella, salmonella, listeria, fungi,yeast, insect cells such as Spodoptera frugiperda (SF9), animal cellssuch as CHO and mouse cells, African green monkey cells such as COS 1,COS 7, BSC 1, BSC 40, and BMT 10, human cells, and plant cells in tissueculture. Preferred host organisms include bacteria such as E. coli andB. subtilis, and mammalian cells in tissue culture.

It should, of course, be understood that not all vectors and expressioncontrol sequences will function equally well to express the nucleotidesequences/DNA sequences of this invention. Neither will all hostsfunction equally well with the same expression system. However, one ofskill in the art may make a selection among these vectors, expressioncontrol sequences and hosts without undue experimentation and withoutdeparting from the scope of this invention. For example, in selecting avector, the host must be considered because the vector must replicate init. The vector's copy number, the ability to control that copy number,and the expression of any other proteins encoded by the vector, such asantibiotic markers, should also be considered. In selecting anexpression control sequence, a variety of factors should also beconsidered. These include, for example, the relative strength of thesequence, its controllability, and its compatibility with the nucleotidesequences/DNA sequences of this invention, particularly as regardspotential secondary structures. Unicellular hosts should be selected byconsideration of their compatibility with the chosen vector, thetoxicity of the product coded for by the nucleotide sequences/DNAsequences of this invention, their secretion characteristics, theirability to fold the protein correctly, their fermentation or culturerequirements, and the ease of purification from them of the productscoded for by the nucleotide sequences/DNA sequences of this invention.Within these parameters, one of skill in the art may select variousvector/expression control sequence/host combinations that will expressthe nucleotide sequences/DNA sequences of this invention on cultivationor in large-scale animal culture.

The polypeptides, i.e. the N-terminal fragments, proteins andimmunogenic fusion protein, encoded by the nucleotide sequences/DNAsequences of this invention may be isolated from the microbial cultureor cell culture and purified using any of a variety of conventionalmethods including: liquid chromatography such as normal or reversedphase, using HPLC, FPLC and the like; affinity chromatography (such aswith inorganic ligands or monoclonal antibodies); ion exchangechromatography, size exclusion chromatography; immobilized metal chelatechromatography; gel electrophoresis; tangential flow filtration and thelike. One of skill in the art may select the most appropriate isolationand purification techniques without departing from the scope of thisinvention.

In addition, the polypeptides, i.e. the N-terminal fragments, proteinsand immunogenic fusion protein of this invention may be generated by anyof several chemical techniques. For example, they may be prepared usingthe solid-phase synthetic technique originally described by R. B.Merrifield (J Am Chem Soc 1963 83:2149-54), or they may be prepared bysynthesis in solution. A summary of peptide synthesis techniques may befound in E. Gross & H. J. Meinhofer, 4 The Peptides: Analysis Synthesis,Biology; Modern Techniques Of Peptide And Amino Acid Analysis, JohnWiley & Sons, (1981); and M. Bodanszky, Principles Of Peptide Synthesis,Springer-Verlag (1984).

An eight aspect of the present invention concerns a method of producingan immunogenic fusion protein comprising the steps of

-   -   a. Providing a host cell according to the seventh aspect of the        present invention,    -   b. Multiplying the host cell,    -   c. Purifying the immunogenic fusion protein according to the        first aspect of the present invention and    -   d. Obtaining the immunogenic fusion protein.

Alternatively the immunogenic fusion protein can be produced in acell-free expression system.

Such systems comprise all essential factors for expression from anappropriate recombinant nucleic acid, operably linked to a promoter thatwill function in that particular system.

A ninth aspect of the present invention concerns a method for preventingor treating an infection caused by a group B Streptococcus whichcomprises administering to an individual an effective amount of avaccine as described herein.

These methods comprise administering to an individual a pharmaceuticallyeffective amount of the vaccine of the invention. There is also,according to the present invention, provided a use of the immunogeniccomposition of the invention for the manufacture of a vaccine forpreventing or treating an infection caused by a group B Streptococcus.

Maternal immunoprophylaxis with a vaccine, for protecting againstinfection to group B Streptococcus both in the mother and in the younginfant, has long been proposed as a potential route.

Thus one embodiment of the method according to the ninth aspect of thepresent invention comprises administering to a human female an effectiveamount of a vaccine as described herein capable of conferring immunityto the infection to an unborn offspring of the human female.

According to this embodiment, the vaccine is administered to anon-pregnant female or to a pregnant female, under conditions of timeand amount sufficient to cause the production of antibodies which serveto protect both the female and a fetus or newborn (via passive transferof antibodies across the placenta).

The terms “preventing or treating” in its various grammatical forms inrelation to the present invention refer to preventing, curing,reversing, attenuating, alleviating, ameliorating, inhibiting,minimizing, suppressing, or halting (1) the deleterious effects of adisorder associated with group B Streptococcus infection, (2) disorderprogression, or (3) disorder causative agent (group B Streptococcus).Further, the terms “preventing or treating” are contemplated to includethe creation of total or partial immunity of the individual to group BStreptococcus infection.

A tenth aspect of the present invention concerns a method for preventingor treating an infection caused by a group B Streptococcus whichcomprises administering to an individual in need thereof an effectiveamount of antibodies elicited from the exposure of a second individualto a vaccine according to one aspect of the invention.

According to this embodiment, resistance to group B Streptococcus isconferred to the individual by passive immunization, i.e., the vaccineis provided to a host (i.e. a human or mammal) volunteer, and theelicited antisera is recovered and directly provided to a recipientsuspected of having an infection caused by a group B Streptococcus. Itis contemplated that such antisera could be administered to a pregnantfemale (at or prior to parturition), under conditions of time and amountsufficient so that the antisera would serve to protect either the fetusor newborn (via passive incorporation of the antibodies across theplacenta).

The vaccine or antisera of the present invention may, thus, be providedeither prior to the onset of infection (so as to prevent or attenuate ananticipated infection) or after the initiation of an actual infection.

The vaccine may be administered to humans or animals, including mammalsand birds, such as rodents (mouse, rat, guinea pig, or rabbit); birds(turkey, hen or chicken); other farm animals (cow, horse, pig orpiglet); pets (dog, cat and other pets); and humans. While many animalsmay be treated with the vaccine of the invention, a preferred individualfor treatment is a human or commercially valuable animal and livestocksuch as fish, e.g. Tilapia, and camels.

The vaccine can be administered to an individual according to methodsknown in the art. Such methods comprise application e.g. parenterally,such as through all routes of injection into or through the skin: e.g.intramuscular, intravenous, intraperitoneal, intradermal, mucosal,submucosal, or subcutaneous. Also, they may be applied by topicalapplication as a drop, spray, gel or ointment to the mucosal epitheliumof the eye, nose, mouth, anus, or vagina, or onto the epidermis of theouter skin at any part of the body. Other possible routes of applicationare by spray, aerosol, or powder application through inhalation via therespiratory tract. In this last case the particle size that is used willdetermine how deep the particles will penetrate into the respiratorytract. Alternatively, application can be via the alimentary route, bycombining with the food, feed or drinking water e.g. as a powder, aliquid, or tablet, or by administration directly into the mouth as a:liquid, a gel, a tablet, or a capsule, or to the anus as a suppository.The vaccine may also be administrated in the form of a DNA vaccine.

Many different techniques exist for the timing of the immunizations. Itis possible to use the compositions of the invention more than once toincrease the levels and diversities of expression of the immunoglobulinrepertoire expressed by the immunized animal. Typically, if multipleimmunizations are given, they will be given one to two months apart.

The term “effective amount” in relation to the present invention refersto that amount which provides a therapeutic effect for a given conditionand administration regimen. This is a predetermined quantity of activematerial calculated to produce a desired therapeutic effect inassociation with the required additives and diluents; i.e., a carrier,or administration vehicle. Further, it is intended to mean an amountsufficient to reduce and most preferably prevent a clinicallysignificant deficit in the activity and response of the host.Alternatively, a therapeutically effective amount is sufficient to causean improvement in a clinically significant condition in a host. As isappreciated by those skilled in the art, the amount of a compound mayvary depending on its specific activity. Suitable dosage amounts maycontain a predetermined quantity of active composition calculated toproduce the desired therapeutic effect in association with the requireddiluents; i.e., carrier, or additive. Further, the dosage to beadministered will vary depending on the active principle or principlesto be used, the age, weight etc of the individual to be treated.

Dose-finding experiments have been done for the previously describedfusion protein disclosed in WO 2008127179 in both mice and humans. Inmice dose-response was seen at doses from approximately 80 ng to 2 μg inthe presence of Alhydrogel, i.e. AlOH, with a plateau reached above 2μg. In humans, 10 ug, 50 μg and 100 μg doses were tested in the absenceor presence of Alhydrogel. For the alhydrogel group, the 10 μg dose wasjust at the top of the dose response curve, and 50 and 250 μg at theplateau. The preferred human doses of the immunogenic fusion proteinaccording to the first aspect of the present invention in the presenceof Alhydrogel is therefore within the range of 1 to 250 μg, preferably10 to 150 μg, preferably 25 to 100 μg or 40 to 80 μg. In the absence ofAlhydrogel, the preferred human doses of the immunogenic fusion proteinaccording to the first aspect of the present invention would be 10 to100 μg, preferably 50 to 500 μg, or preferably 100 to 250 μg.

Generally, the dosage may consist of an initial injection, most probablywith adjuvant, followed most probably by one or maybe more boosterinjections. Preferably, booster injections may be administered at about1 and 6 months after the initial injection.

The invention claimed is:
 1. An immunogenic fusion protein consisting ofan amino acid sequence having at least 90% sequence identity with theamino acid sequence shown in SEQ ID NO:12.
 2. The immunogenic fusionprotein according to claim 1, wherein the amino acid sequence has atleast 95% sequence identity with the amino acid sequence shown in SEQ IDNO:12.
 3. The immunogenic fusion protein according to claim 1, whereinthe amino acid sequence has at least 99% sequence identity with theamino acid sequence shown in SEQ ID NO:12.
 4. The immunogenic fusionprotein according to claim 1, wherein the amino acid sequence consistsof the amino acid sequence shown in SEQ ID NO:12.
 5. An immunogeniccomposition comprising the immunogenic fusion protein according to claim1 and a pharmaceutically acceptable vehicle.
 6. The immunogeniccomposition according to claim 5, further comprising aluminiumhydroxide.